title: Development of a high-throughput AlphaScreen assay for modulators of synapsin I phosphorylation in primary neurons.
abstract: Alterations in synaptic transmission have been implicated in a number of psychiatric and neurological disorders. The discovery of small-molecule modulators of proteins that regulate neurotransmission represents a novel therapeutic strategy for these diseases. However, high-throughput screening (HTS) approaches in primary neurons have been limited by challenges in preparing and applying primary neuronal cultures under conditions required for generating sufficiently robust and sensitive HTS assays. Synapsin I is an abundant presynaptic protein that plays a critical role in neurotransmission through tethering synaptic vesicles to the actin cytoskeleton. It has several phosphorylation sites that regulate its modulation of synaptic vesicle trafficking and, therefore, the efficacy of synaptic transmission. Here, we describe the development of a rapid, sensitive, and homogeneous assay to detect phospho-synapsin I (pSYN1) in primary cortical neurons in 384-well plates using AlphaScreen technology. From results of a pilot screening campaign, we show that the assay can identify compounds that modulate synapsin I phosphorylation via multiple signaling pathways. The implementation of the AlphaScreen pSYN1 assay and future development of additional primary neuronal HTS assays provides an attractive approach for discovery of novel classes of therapeutic candidates for a variety of CNS disorders.
authors.affiliation: 1Galenea Corporation, Wakefield, MA, USA.
authors.forename: Betty
authors.initials: B
authors.lastname: Chan
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Cottrell
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: Kelley C
authors.initials: KC
authors.lastname: Larson
authors.forename: Crystle J
authors.initials: CJ
authors.lastname: Ashford
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Levenson
authors.forename: Pascal
authors.initials: P
authors.lastname: Laeng
authors.forename: David J
authors.initials: DJ
authors.lastname: Gerber
authors.forename: Jianping
authors.initials: J
authors.lastname: Song
mesh_terms: D001921:Brain / Q000378:metabolism / Q000473:pathology*; D002478:Cells, Cultured; D057166:High-Throughput Screening Assays; D006801:Humans; D009422:Nervous System Diseases / Q000188:drug therapy / Q000378:metabolism; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D015398:Signal Transduction; D016704:Synapsins / Q000302:isolation & purification* / Q000378:metabolism; D009435:Synaptic Transmission / Q000235:genetics*; D013572:Synaptic Vesicles / Q000378:metabolism
chemical_list: D016704:Synapsins
keywords: AlphaScreen; high-throughput screening; synapsin